When a protein is produced in Gram-negative bacteria such as E. coli, a protein of interest may often be secreted into a space called periplasm between the inner membrane (the cell membrane) and the cell wall by attaching a signal sequence at the N-terminal of said protein. However, efficiency of transmitting a protein of interest into periplasm through the inner membrane varies depending on a combination of a signal sequence and a protein of interest and there is no known approach for affording always high transmission. It is known that a proteolytic enzyme (protease) is also secreted into periplasm of E. coli and thus a protein of interest secreted into periplasm may be subject to proteolysis.
On the other hand, when a protein of interest is secreted into periplasm under denaturation condition, the protein may sometimes form a structure called an inclusion body. It is said that a protein of interest incorporated into an inclusion body is not likely to be subject to proteolysis by protease. Besides, since an inclusion body incorporates a high concentration of a protein of interest, it may advantageously be used in view of efficiency of purification and high-yield recovery. However, there is no known approach at present for expressing a protein of interest in Gram-negative bacteria such as E. coli and forming efficiently an inclusion body by e.g. modifying a signal peptide.
Ibrahim et al. showed that, by substituting leucine with proline at P8 in a hydrophobic core region of a signal in endotoxin subunit B of E. coli in cell-free protein synthesizing system using wheat germ, cleavage of a signal sequence is prohibited and a synthetic rate increased two-fold (cf. e.g. Non-patent reference 1). However, since the results of this process are outcome of a simplified expression system with cell-free system, the process would not necessarily be applicable to an expression system with living cells such as E. coli. In addition, a protein synthesized by this process was soluble and did not form an inclusion body.
There is another report that, when mutation (mutation from leucine to proline at P17) occurs spontaneously in a signal sequence of a ribose-binding protein in E. coli, cleavage of a signal sequence is prohibited and a soluble ribose-binding protein precursor is accumulated in cytoplasm, and that its expression level was so small that detection with labeling using radioisotope was necessary and was equivalent to that of a wild-type protein before mutation (cf. e.g. Non-patent reference 2). As such, an effect of mutation in a signal sequence from prokaryotic cells on expression of an inclusion body in E. coli has not well been elucidated and, in particular, an effect of the mutation on expression of a structural gene from eukaryotic cells is utterly not known.
When a recombinant cell with a heterologous gene incorporated therein is used for production of a protein, if a selection medium containing antibiotics is not used, it may occur that a recombinant cell excludes the incorporated gene during proliferation to lose the character of said gene and such a recombinant cell may predominantly proliferate to result in reduced efficiency in production of a protein. Thus, when a gene (e.g. an expression plasmid) is introduced into a host cell, such a gene that affords resistance to antibiotics used for selection through degradation or modification of the antibiotics is also simultaneously introduced into the host cell and cell culture is performed in a selection medium containing antibiotics toxic to a host cell not conveying said gene (such as e.g. ampicillin and tetracycline) to allow for selection of those recombinant cells that carry the gene and for restraint of generation of those recombinant cells that exclude the introduced gene.
However, antibiotics may be toxic to living organisms and cause drug hypersensitivity (allergy). Accordingly, for the manufacture and sale of a medicament, an animal drug or a food stuff using a recombinant cell, their contamination with antibiotics needs strictly be controlled. As such, a constitution and a method are desired where an introduced gene may be maintained without selection with antibiotics.
MMP-7 is among matrix metalloproteases (hereinafter also referred to as “MMP”) belonging to a zinc-type metalloprotease family where a zinc molecule is present at the active site (cf. e.g. Non-patent reference 3). MMP is produced as a precursor. The precursor is processed to cleave a signal sequence when extracellularly secreted and then processed to cleave a pro sequence to generate an active form. It is reported that extracellularly secreted MMP is involved in metabolism of extracellular matrix whereas MMP-7 is mainly secreted from cancer cells and is involved in infiltration and metastasis of cancer (cf. e.g. Non-patent reference 4). MMP-7, not possessing a hinge domain and a hemopexin-like domain unlike other MMPs, consists of the minimum molecular unit among MMP and its substrate is collagen and components that constitute extracellular matrix such as fibronectin, vitronectin, laminin and aggrecan.
It is assumed that MMP-7 may be involved in natural resorption on of hernia disk viewing that its substrate is aggrecan, a principal component of cartilage, and that macrophages from samples from surgical operation of disk herniation express MMP-7 (cf. e.g. Non-patent reference 5). Subsequently, Haro et al. observed reduction in a volume of the nucleus pulposus in the intervertebral disks after administration of MMP-7 into the intervertebral disks of hernial dogs and showed possible use of MMP-7 as a medicament for disk herniation (cf. e.g. Non-patent reference 6). Development of MMP-7 for medical usage is desired. However, MMP-7 occurs only in a trace amount in the living body and thus its isolation and purification from the living body is extremely difficult. Besides, when living material is used, there will be concern for safety problem such as potential viral contamination. Although MMP-7 may be obtained from cancer cells, it is not preferable to use cancer cells as a source for production (cf. e.g. Non-patent reference 7).
For solving the above problems, an attempt to obtain MMP-7 by a genetic recombination technique has been made. For a system using animal cells, there is a report by Barnett et al. that MMP-7 is expressed in CHO cells (cf. e.g. Non-patent reference 8). However, an expression level of MMP-7 is as low as around several mg/L and thus the reported system is not actually suited for production of a medicament. It is also reported that a nucleic acid fragment in which a nucleotide sequence coding for a signal sequence of alkali phosphatase and a gene sequence of proMMP-7 with optimization to codon usage in E. coli are bound to each other is used to allow for expression of soluble MMP-7 at 34° C. and expression of insoluble MMP-7 at 42° C. (cf. e.g. Patent reference 1).